Calcium and Heme Induce Distinct Conformational States of the Human BK (Slo1) Channel Gating Ring

The human BK channel gating ring (GR) is the site of action of numerous signaling molecules such as Ca2+ and heme, which ultimately modulate pore open probability. However, the precise nature of these structural rearrangements remains elusive. Recently, we demonstrated that heme-induced conformational changes in the GR are not additive to Ca2+-induced structural rearrangements (Yusifov et al., BPS meeting 2010), posing a question about the extent of similarity in the mechanical operation of the GR upon Ca2+ or heme binding. To address this question, we analyzed the conformational states of apo-GR and Ca2+- or heme-bound GR by characterizing dynamic fluorescence quenching by acrylamide, a quencher of the excited state of accessible native tryptophan (Trp) residues. The time-resolved Trp fluorescence (λex=296 nm, λem=340 nm) of apo-GR and ligand-bound GR with the progressive addition of acrylamide (up to 0.4 M) was recorded. Acrylamide addition resulted in the reduction of the mean Trp fluorescence lifetime (calculated from three-exponential iterative fittings) of apo-GR (τ0=2.74±0.05ns to τ=1.5±0.1ns), Ca2+-bound-GR (τ0=2.1±0.02ns to τ=1.27±0.021ns) and heme-bound-GR (τ0=2.1±0.1ns to τ=1.0±0.05ns). The acrylamide-Trp bimolecular quenching constants (kq) were extracted from Stern-Volmer plots of τ0/τ vs. [acrylamide]. Supplementation of saturating [Ca2+] (35 μM) or [heme] (700 nM) increased kq from 8.40×108 M−1s−1 (apo-GR) to 1.19×109 M−1s−1 and 1.95×109 M−1s−1, respectively. The increased efficiency of Trp quenching by acrylamide in the ligand-bound GR implied that native Trp residues were more accessible to the quencher, supporting ligand-induced conformational changes. Since different kq values were derived from the two ligands, it is likely that Ca2+ and heme binding induced the GR to adopt different conformational states. We propose that the two ligands can modulate BK open probability through distinct intramolecular signal transduction pathways.